| BackgroudThe present studies show that occurance of malignant tumors is closely related to the block or disorder of cell differentiation. The conventional therapies for treatment of malignant disorders, including radiotherapy, chemotherapy, molecular targeting and biological therapy are all base on the concept of eliminating tumor cells, of which will damage the nomal cells as well in the process. More and more studies indicate that induced by in vitro or in vivo agent, variant marks of the malignant tumor cells including those of the morphology, biology and biochemistry, evolve to normal differentiation and the tumor cells even transfer to complete or near complete normal cells. This phenomenon is termed tumor cell redifferentiation. Therefore, the inducing differentiation therapy may offer a new developing direction in the strategy of malignant tumor treatment.Hodgkin lymphoma is the malignant tumor of lymphoid tissues firstly described by Dr. Thomas Hodgkin in 1832. The classic Hodgkin lymphoma (cHL) which is the major type of HL, accounts for about 95% of the cases. The cHL is characterized by the rare so-called Hodgkin/Reed-Sternberg (H/RS) tumour cells (< 1%) embedded in the background of predominant recative inflammatory cells. For more than 170 years, the nature and mechanism of development of H/RS cells remained illusive to be interpreted in medical circle.Kuppers et al. picked out single H/RS cells from the biopsy specimen through microdissection technique, and single-cell PCR analysis of the immunoglobin (Ig) gene rearrangement of these cells proved that overwhelming majority of the H/RS originated from the pre-apoptotic germinal centre B cells called crippled B-cells in lymph organs. The H/RS cells are characterized by the loss of B-cell phenotype and evidence of abortive plasma cell differentiation. The key question in this study is to find out the pivotal targets that lead to differentiation block during generation of H/RS cells, and restore H/RS from B-cell makers through regulating these "switch" target back to the normal direction of B cell differentiation.It has been reported that the morphology and immunophenotype of human B lymphoma cell lines (BJAB, IM9) transfected with antisense CD99 expression plasmid were similar with H/RS cells, suggesting that downregulation of CD99 expression is a key molecular event in the generation of H/RS cells. The transcription factor PRDM1, a "switch"of inducing B cells differentiation towards plasma cells, is a master regulator of plasma cell differentiation. The present studies suggest that PRDM1 is an important anti-oncogene in cHL, the functional inactivation of which may play a role in the pathogenesis of cHL by blocking the B cells differentiation towards plasma cells. H/RS cells have the potential of early plasma-cell differentiation, and induction H/RS cells towards terminal B cells must be based on increase of PRDM1 expression, a "switch" of plasma-cell differentiation. Recent studies have found that miRNAs play an important role in B cell differentiation and malignant B-cell lymphomas. Nie et al have confiremed that PRDM1 which is a direct target of miR-9, is negatively regulated by miR-9 in the cHL cell line of L428 by luciferase reporter experiments. Thus, miR-9 may be another important target for differentiated arrest of H/RS cells.Our research group have previously constructed subseries of L428 cell line with stable overexpression of CD99 gene, named "L428-CD99". On this basis, we plan to identify the sub-cell line of L428-CD99, and detect the effect of upregulation of CD99 on cell morphology, the biological characteristics and differentiation-related cell phenotype in L428 cells, and explore whether overexpression of CD99 can be restored from B-cell phenotype, and induce redifferentiation of H/RS cells by upregulation of PRDM1, and verify expression of miR-9 in H/RS cells and its regulation on target PRDM1, and explore the possibility of induction of redifferentiation of H/RS cells, and provide reference for further elaborating mechanisms of generation and development of H/RS cells.ObjectiveThis study will be carried out in four parts:1. The expression level of CD99 and PRDM1 in cHL tissues and cell linesDetecting differences of CD99 and PRDM1 expression in reactive tissues of lymph node hyperplasia (RH), cHL tissues and cell lines, B-cell original and T-cell original lymphoma cell lines.2. CD99 regulation on PRDM1 inducing redifferentiation of H/RS cellsExploring the interaction between CD99 and PRDM1, and detecting the effect of upregulation of CD99 gene on diagnosis markers of cHL, B-cell differentiation-related phenotype and plasma-cell differentiation markers in L428 cells, revealing the role of CD99 gene in B cell differentiation and H/RS cell generation and transformation.3. The expression of miR-9 in H/RS cells and its regulation on target PRDM1Detecting miR-9 expression in cHL cell line quantitatively and positionally, and exploring the regulation of miR-9 on target PRDM1 by transient knockdown of miR-9 expression.4. Preliminary investigations of regulation between CD99 and miR-9Preliminary experiments and bioinformatics analysis of regulatory nets between miR-9 and PRDM1, so as to explore regulatory relation among CD99, miR-9 and PRDM1.Contents and methods1. CD99 and PRDM1 expression in cHL tissues and cell linesThe specimens of cHL cases and reactive lymph node hyperplasia were collected and analyzed by immunohistochemistry of CD99 and PRDM1. CD99 and PRDM1 protein expression were further measured by Western blot and immunocytochemistry in six B-cell origin and three T-cell origin lymphoma cell lines.2. CD99 regulation on PRDM1 inducing redifferentiation of H/RS cells(1) Identification of L428-CD99 sub-cell lineGFP expression was observed by fluorescence microscope in sub-cell line of L428-CD99 which was previously stablely transfeced by CD99 gene lentivirus vector. The mRNA and protein expression of CD99 was measured by real-time PCR, Western blot and confocal microscope.(2) The effect of CD99 overexpression on morphology and biological characteristics of cHL cell line L428.The effect of CD99 overexpression on proliferation, celluar size and cytoskeletal proteins was detected by MTT, HE staining and phalloidin staining.(3) The effect of CD99 overexpression on differentiation-related proteins in L428 cellsThe PRDM1 protein expression was detected by Western blot and confocal microscope after stable tansfection of CD99 gene. The diognosis markers of cHL (CD30, CD15), B cell differentiation-related proteins (CD10, CD19, CD20, CD45, CD79a, BCL6, PAX5 and MUM1) and plasma cell markers (CD38 and CD138) were detected by immunocytochemistry and flow cytometry.3. The expression of miR-9 in H/RS cells and its regulation on target PRDM1(1) Detection of miR-9 expression in L428 cellsMagnetic separation of CD19+ B lymphocytes was performed from normal lymph nodes as a control. The miR-9 expression was detected by real-time PCR and in situ hybridization (ISH) in isolated CD19+ B-cell subsets and eight lymphoma cell lines including L428 cells.(2) The effect of transient interference of miR-9 on PRDM1 protein expression and cellular proliferation in L428 cellsTransfection efficiency was analyzed by fluorescence microscope, and interference efficiency was detected by real-time PCR after transient transfection of miR-9 antisense oligonucleotide. The PRDM1 protein expression was detected by Western blot and confocal microscope after knockdown of miR-9. The effect of miR-9 antisense oligonucleotide on proliferation of L428 cells was detected by MTT assay.4. Preliminary investigations of regulation between CD99 and miR-9The expression of miR-9 was detected by real-time PCR after upregulation of CD99 gene. The expression of CD99 was detected by real-time PCR and Western blot after interference of miR-9. Bioinformatics analysis of signal regulatory pathway between miR-9 and PRDM1 was performed.Results1. CD99 and PRDM1 expression in cHL tissues and cell lines (1) CD99 was mainly expressed in mantle zone B cells and interfollicular area of reactive lymphoid hyperplasia tissues (RH) and PRDM1 was expressed in light zones of germinal centers (GC). The positivity for CD99 was found in 1 of the 62 cHL cases examined, and PRDM1 expression was not found in H/RS cells of all cHL cases.(2) CD99 and PRDM1 were highly expressed in multiple myeloma (MM) cell line RPMI-8226, and lowly or absently expressed in L428 cell and other B-cell origin of lymphoma cell lines.2. CD99 regulation on PRDM1 inducing redifferentiation of H/RS cells(1) The strong green fluorescence was observed in L428-CD99 sub-cell line under fluorescence microscope. As indicated, CD99-upregulated L428 cells showed increased expression of CD99 gene and protein expression compared with mock and empty vecter groups using real-time PCR, Western blot and confocal microscopy analysis.(2) MTT assay indicated that CD99-overexpressed L428 cells showed reduced growth compared with controls, with significant difference (F= 305.917, P= 0.000). H&E staining analysis showed that the ratio of 20+ uM cells occupying the total cell number in L428-CD99 cells is (50.187±2.518)%, compared with that of (77.588±5.580)% in control groups, with significant difference (Z=- 2.611, P= 0.008). When cells were stained with phalloidin-FITC to label filamentous actin, we clearly observed the disappearance of filopodia and thinningz of cortical filamentous actin in CD99-transfected L428 cells compared with control cells.(3) Western blot and confocal microscopy analysis showed that L428 cells with overexpression of CD99 showed a concomitant increasing expression of PRDM1 protein compared with mock and empty vecter groups. The L428 cells transfected with CD99 gene showed the decrease of CD30, CD 15 and MUM1 expression, and increase of CD10, CD19, CD79a, BCL-6, PAX5 and CD38 expression. There was no significant change for CD20 and CD138 by immunocytochemistry and flow cytometry analysis, compared with those of control cells.3. The expression of miR-9 in H/RS cells and its regulation on target PRDM1(1) The results of qRT-PCR showed that expression of miR-9 in L428 cells is markedly higher than those of isolated CD19+ B-cell subsets and other lymphoma cell lines, with significant difference (P< 0.05). In situ hybridization showed that miR-9 was extensively localized in the cytoplasm. A strong and uniform cytoplasmic signal was observed in cHL cell line, compared with scattered signal in DLBCL and Burkitt's lymphoma cell lines, while no signal was detected in T-cell origin of KARPAS-299 and Jurkat cells.(2) The results of real-time PCR showed that enforced miR-9 inhibitor resulted in an almost 50% decrease of miR-9 expression in L428 cells (t=-5.208, P= 0.035).(3) The results of Western blot showed that the L428 cells transfected with miR-9 inhibitor showed an increase of PRDM1 expression, compared with those of mock and control grpups.(4) No remarkable differences in cell growth were observed among the mock, negative control and miR-9 inhibitor groups (F= 1.069, P= 0.350).4. Preliminary investigations of regulation between CD99 and miR-9(1) The results of real-time PCR showed that expression of miR-9 was significantly downregulated by introduction of CD99 into L428 cells (t= 282.071, P= 0.000).(2) The CD99 expression remained generally unaffected in cells transfected with miR-9 inhibitor, compared with those of mock and negative control groups by real-time PCR (P= 0.719, P= 1.000). The same result was also confirmed by western blot.(3) Bioinformatics analysis showed that CD99 may regulate miR-9 expression, through KPNB1-(SMAD3, SMAD4)-MYC signal pathway.Conclusion1. Both of CD99 and PRDM1 were lowly expressed or absent in cHL tissues and cell lines.2. The overexpressed CD99 gene lentivirus vector in L428-CD99 sub-cell line is stable. The overexpressed CD99 gene leads to upregulation of CD99 mRNA and protein expression, reduced cell growth, decrease of cellular size and reorganization of actin cytoskeleton in L428 cells. Furthermore, upregulation of CD99 leads to a decrease in cHL diagnosis marker CD30 and CD 15, restoration of B-cell makers, and increase in plasma cell differentiation key factor PRDM1 protein along with plasma cell marker CD38, suggesting characteristics of pre-plasmablast cells.3. MiR-9 is specificly overexpressed in L428 cells and may negatively regulate the expression of PRDM1.4. CD99 negatively regulate miR-9, while miR-9 can not react up on CD99 expression. CD99 may regulate PRDM1 expression through controlling miR-9 expression.Discoveries and innovations1. Our results suggest that generation of H/RS is closely associated with B-cell differentiation block and disorders, reveal that CD99, miR-9 and PRDM1 is tightly related to generation and development of cHL.2. We have preliminarily revealed that CD99 may regulate miR-9 to mediate PRDM1 expression, which induces restoration of B-cell makers and differentiation towards terminal B cells. This research provids the theoretical basis and accurate target for differentiation therapy, with original creativity. |
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